CN112587719A

CN112587719A - Antibacterial hemostatic membrane and preparation method and application thereof

Info

Publication number: CN112587719A
Application number: CN202110059037.1A
Authority: CN
Inventors: 叶飞; 金甲; 王磊; 徐�明; 张文平; 舒建洪; 吕正兵
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2021-01-17
Filing date: 2021-01-17
Publication date: 2021-04-02

Abstract

The invention relates to an antibacterial hemostatic membrane and preparation and application thereof, wherein the antibacterial hemostatic membrane takes sodium alginate, carboxymethyl chitosan, collagen and berberine hydrochloride as raw materials, and a biological composite hemostatic membrane is prepared by adding a humectant and atomizing and adding calcium chloride small drops as a cross-linking agent; good biocompatibility, no blood dissolution, rapid and efficient degradation, and no influence on wound healing.

Description

Antibacterial hemostatic membrane and preparation method and application thereof

Technical Field

The invention relates to an antibacterial hemostatic membrane containing berberine hydrochloride, a preparation method and related applications thereof.

Background

The skin is the first barrier of human body to resist external stimulation and injury, and people can be injured due to mechanical injury, burn, operation, war and the like all the time, and the wounds are often accompanied by bleeding and bacterial infection. Loss of blood can lead to a series of systemic reactions in the body, even shock and death, with nearly half of trauma victims being caused by uncontrolled bleeding. Wound infection is a pathological reaction in which pathogenic microorganisms invade the human body through wounds, grow and multiply in the body, and cause local changes or systemic poisoning in the human body. Therefore, the key to preventing bleeding death is safe and effective rapid hemostasis.

With the development of the times, the materials and the way of stopping bleeding are diversified. Ancient China applied the mashed herbaceous plant fiber to the wound, and then bandaged with cloth strips for compression hemostasis; the ancient Egyptian uses a mixture of beeswax, barley and grease for hemostasis; ancient indians used a mixture of red sand and animal fur for hemostasis, and the natural drawbacks of such mixtures are that they do not degrade naturally and are prone to infection and the like. In western countries before the middle century, there are methods for stopping bleeding by burning wounds, and carbonization of the wounds can cause rapid scabbing and high-temperature sterilization can be effectively performed. Although the hemostatic effect and the anti-infection effect are quite good, the hemostatic effect and the anti-infection effect cause great pain to people when the hemostatic agent is used, and scars are left after the wounds heal. In recent times in China, along with the improvement of cognition of people on the medical hemostatic, a plurality of trauma specific medicines such as incised wound medicines, Yunnan white medicines and the like are added in the formula of the traditional Chinese medicine hemostatic. After the western industrial revolution, zeolite has also once become a new favorite for military and industrial medical hemostasis. The rapid development of scientific and technological civilization, the deep research on the hemostatic mechanism, improves the requirements on hemostatic materials, and the alpha-cyanoacrylate (PLA) and polyethylene glycol (PEG) materials synthesized by artificial chemistry can rapidly form high-viscosity colloid to block blood vessels, thereby achieving the effect of effective hemostasis. PLA and PEG hemostatic materials are widely used in surgical procedures to stop bleeding while improving the safety of products by improving the preparation process. At present, more researchers invest a great deal of energy and financial resources in the research of biological composite hemostatic materials. Firstly, biological materials with excellent biocompatibility, such as starch, oxidized cellulose, alginate, chitosan, collagen, fibrin, thrombin and the like, are adopted as raw materials for preparation, so that toxic and side effects are reduced from the source. Secondly, various special materials are added to improve the hemostatic effect in practical application or increase the anti-infection capacity, such as composite nano silver, graphene, kaolin, montmorillonite, zeolite and the like. Compared with inorganic mineral hemostatic materials or polymer synthetic hemostatic materials, the biological composite hemostatic material has better biocompatibility, lower cytotoxicity and easier degradation in vivo; compared with natural polymer hemostatic materials, the biological composite hemostatic material has better hemostatic effect and anti-infection capability.

Chinese patent CN 107281539A discloses a preparation method of a soluble paper-like alginate composite hemostatic membrane, which is formed by processing and molding by electrostatic spinning equipment. The soluble hemostatic membrane is prepared from alginate and polyvinyl alcohol, has a porous reticular structure and a higher surface area, can quickly form gel to accelerate the hemostatic process by interacting with blood of a wound, and simultaneously calcium ions in the hemostatic membrane material release a wound surface to start a blood coagulation factor so as to achieve dual hemostatic effects of gel hemostasis and calcium ion blood coagulation. The invention mainly applies the hemostatic effect of alginate, and although the process is simple and the biological safety of the raw materials is high, the hemostatic effect is limited, the anti-infection capability is lacked, and the quick hemostatic environment is difficult to apply.

Chinese patent CN 109260507A discloses a high liquid absorption silk fibroin hemostatic membrane and a preparation method thereof, wherein the hemostatic membrane comprises polyoxyethylene, silk fibroin, sodium alginate and nano silicon dioxide, has a good hemostatic effect, can absorb a large amount of wound exudate, and has good air permeability and biocompatibility. The porous membrane material prepared by the method has certain flexibility and tensile property, provides few selectable and replaceable schemes, and has certain limitation on the source of raw materials of the product in large-scale application.

Chinese patent CN 110755672A discloses an antibacterial hemostatic sponge and a preparation method thereof, wherein the main raw materials are polylactic acid, oxidized cellulose and chitosan, and the problems of large wound bleeding and easy infection are solved. The antibacterial hemostatic sponge has good biocompatibility, good compressibility, softness and hydrophilicity, is suitable for wounds with large bleeding amount, but the chitosan with ultrahigh deacetylation degree and the foaming agent in the raw materials have high supercritical carbon dioxide cost, and the preparation process is complicated and is not beneficial to commercial production.

Chinese patent CN 109568643A discloses a preparation method and application of berberine-containing antibacterial hemostatic microspheres, which mainly comprise carboxymethyl chitosan, sodium alginate, collagen and berberine. Is characterized in that the antibacterial and adhesive characteristics of the berberine are utilized, the antibacterial effect of the material is improved, and the hemostatic effect is further enhanced. However, the hemostatic microspheres are difficult to deal with large-area wounds, and blood flow easily causes the loss of the microsphere material, so that the contact between the hemostatic microspheres and the wound bleeding is reduced, the hemostatic performance of the hemostatic microspheres is affected, and other auxiliary materials are required to be fixed, so as to ensure that the hemostatic effect of the hemostatic microspheres is better exerted. In addition, the water content of the microspheres is very low, the swelling capacity of the material is effectively improved, and the degradation rate is also slowed down.

An ideal hemostatic material should have essentially the following characteristics: rapid hemostasis, anti-infection ability, no influence or promotion on wound healing, good biocompatibility, easy degradation, promotion of blood coagulation, prevention of anticoagulant drug effect, low cost, mass production and easy storage.

Therefore, the invention prepares the novel composite biological antibacterial hemostatic membrane by the composite crosslinking reaction of several natural biomass materials of sodium alginate, carboxymethyl chitosan, collagen and berberine hydrochloride. It is hoped to prepare a hemostatic agent which can be degraded and absorbed rapidly, and release the antibacterial component berberine hydrochloride in the degradation process, so as to achieve the purpose of preventing wound infection. Meanwhile, the berberine hydrochloride improves the adhesion between the material and wound tissues, adjusts the crosslinking degree of the material, accelerates the swelling degradation, effectively aggregates and concentrates platelets and blood coagulation factors, and accelerates the Ca²⁺And the release of berberine cation can improve the hemostatic effect in multiple aspects through adhesion, imbibition and blood coagulation cascade.

Disclosure of Invention

One object of the invention is to provide an antibacterial hemostatic membrane material containing berberine hydrochloride: the hemostatic gel comprises sodium alginate, carboxymethyl chitosan, collagen and berberine hydrochloride, and can rapidly stop bleeding of a large area of local wounds and prevent bacterial infection of the wounds; good biocompatibility and no blood dissolution; meanwhile, the hemostatic film agent is adopted, so that more efficient degradation can be achieved under the condition of further reducing the usage amount of raw materials, and wound healing is not affected.

The antibacterial hemostatic membrane comprises sodium alginate, carboxymethyl chitosan, collagen and berberine hydrochloride, and is characterized in that the average thickness of the membrane is 0.1-2 mm.

Further preferably, the average thickness of the film is 1 to 2 mm.

In the antibacterial hemostatic membrane, the mass ratio of sodium alginate to carboxymethyl chitosan to collagen is as follows: 2-3: 0.1-1: 0.05-0.5.

In the antibacterial hemostatic membrane, the mass ratio of sodium alginate, carboxymethyl chitosan, collagen and berberine hydrochloride is as follows: 2-3: 0.1-1: 0.05-0.5: 0.01-0.5.

In the antibacterial hemostatic membrane, the mass ratio of carboxymethyl chitosan, sodium alginate, collagen and berberine hydrochloride is as follows: 2:1:0.05: 0.03-0.3.

The antibacterial hemostatic membrane further comprises a humectant and a cross-linking agent, wherein the humectant is glycerol, and the cross-linking agent is calcium chloride.

Further preferably, the mass ratio of the sodium alginate to the carboxymethyl chitosan to the collagen to the berberine hydrochloride is as follows: 2:1:0.05: 0.03-0.27, particularly preferably 2:1:0.05: 0.09.

The invention also aims to provide a preparation method of the antibacterial and hemostatic membrane containing berberine hydrochloride, which comprises the following specific steps:

(1) preparing a matrix solution: weighing sodium alginate, carboxymethyl chitosan, collagen and berberine hydrochloride, and adding the components according to the mass-volume ratio of 1: 10-100 parts of distilled water, and magnetically stirring the mixture at normal temperature to form a gelatinous aqueous solution;

(2) adding a humectant: adding glycerol into the prepared matrix solution, and slowly stirring at a uniform speed, wherein the volume ratio or mass ratio of the humectant to the matrix solution is 0.1-3: 10 (ml/ml or g/g);

(3) and (3) crosslinking: adding a cross-linking agent, wherein the volume ratio or the mass ratio of the cross-linking agent to the matrix liquid is 0.5-10: 10 ml/ml or g/g; the reaction time is 12-48 h, the reaction temperature is 4-65 ℃, standing and drying are carried out, and after the reaction is finished, discharging is carried out;

(4) storage: cutting a template with plastic into square films of 10 cm × 10 cm, packaging with aluminum paper, weighing, sealing, and storing in a refrigerator at 4 deg.C.

In the preparation method of the antibacterial and hemostatic membrane containing berberine hydrochloride, preferably, the cross-linking agent in the step (3) is 2% atomized calcium chloride water solution.

The invention also aims to provide application of the antibacterial hemostatic membrane containing berberine hydrochloride in preparation of hemostatic materials.

The invention discloses the effect of the composite antibacterial hemostatic membrane in the application of SD rat tail-cutting hemostasis.

The action mechanism of the invention is as follows: the antibacterial hemostatic membrane containing berberine hydrochloride is a sterile, compact and surface-wrinkled film. The hemostatic membrane has certain water absorption performance, and can activate the blood platelet and the blood coagulation factor, accelerate the blood coagulation cascade process and the blood coagulation effect by concentrating blood and increasing the concentration of the blood platelet and the blood coagulation factor, and meanwhile, the viscosity of the material is improved by using berberine hydrochloride, the crosslinking degree of the material is adjusted, and the hemostasis is further accelerated; degradation experiments show that the material is rapidly degraded and can be degraded within 2 hours; the hemostatic membrane is added with berberine hydrochloride as a main antibacterial component, which influences the active transport and respiration of bacteria, so that the growth of the bacteria is inhibited and even killed, and antibacterial experiments show that the hemostatic membrane has an obvious antibacterial effect and has a more obvious antibacterial effect on gram-positive bacteria; the rat tail-cutting hemostasis experiment shows that the hemostasis is rapid; hemolysis and cytotoxicity experiments prove that the biocompatibility is excellent.

The antibacterial hemostatic film has the following beneficial effects: 1. the hemostatic membrane of the invention has short hemostatic time: the hemostasis is generally completed within 1-2 minutes, and the hemostatic bag is suitable for large-area wound hemostasis; 2. the raw material resources of the invention are rich, the raw material consumption is less, the value is low, and the invention provides a foundation for commercialization; 3. the bacteriostatic action is strong, and the wound infection is prevented; 4. the degradation speed is higher, and the degradation is carried out within 2 h; 5. the use is convenient: covering the hemostatic membrane and fixing the wound bleeding part; 6. good biocompatibility, no blood dissolution and no toxicity. Therefore, the antibacterial hemostatic membrane is an ideal hemostatic agent and is suitable for various bleeding conditions caused by minimally invasive surgeries and daily minor wounds.

Drawings

FIG. 1: example 1 scanning electron micrograph of hemostatic film product;

FIG. 2 is a drawing: examples 2-5 scanning electron micrographs of hemostatic film products;

FIG. 3: examples 1-5 graphs of blood hemolysis experiments on hemostatic membranes;

FIG. 4 is a drawing: examples 1-5 graphs of the results of the hemostatic membrane cytotoxicity experiments;

FIG. 5: the curve of the viscosity of the antibacterial hemostatic membrane along with the change of temperature;

FIG. 6: a curve graph of the storage modulus G' of the antibacterial hemostatic membrane along with the change of strain;

FIG. 7: the storage modulus G' of the antibacterial hemostatic membrane changes with frequency.

Detailed Description

Example 1

1. Preparing a matrix solution: 6g of sodium alginate, 3 g of carboxymethyl chitosan, 0.15 g of collagen and 45 mL of glycerol are respectively added into 555 m1 of water for dissolution, and the mixture is kept stand for 12 hours at normal temperature for removing bubbles in the solution, so that 3 parts of matrix solution is prepared.

2. And (3) crosslinking: preparing 60 mL of 2% calcium chloride solution, slowly pouring the prepared matrix solution into an aluminum alloy container, uniformly spraying calcium chloride aqueous solution by using a spray can, carrying out crosslinking reaction, standing for 15 min, transferring to a 37 ℃ incubator, and standing for 48 h.

3. Storage: a template is taken from plastic, a plurality of square thin films with the thickness of 1-2mm and the size of 10 cm multiplied by 10 cm are cut, the square thin films are packaged by aluminum paper, weighed and sealed, and the square thin films are stored in a refrigerator at 4 ℃.

Example 2

1. Preparing a matrix solution: respectively adding 8 g of sodium alginate, 4 g of carboxymethyl chitosan, 0.2 g of collagen, 0.12g of berberine hydrochloride and 60 mL of glycerol into 740 m1 of water for dissolving, standing for 12 h at normal temperature for removing bubbles in the solution, and preparing 4 parts of matrix solution.

2. And (3) crosslinking: preparing 80 mL of 2% calcium chloride solution, slowly pouring the prepared matrix solution into an aluminum alloy container, uniformly spraying calcium chloride aqueous solution by using a spray can, carrying out crosslinking reaction, standing for 15 min, transferring to a 37 ℃ incubator, and standing for 48 h.

Example 3

1. Preparing a matrix solution: respectively adding 8 g of sodium alginate, 4 g of carboxymethyl chitosan, 0.2 g of collagen, 0.36g of berberine hydrochloride and 60 mL of glycerol into 740 m1 of water for dissolving, standing for 12 h at normal temperature for removing bubbles in the solution, and preparing 4 parts of matrix solution.

Example 4

1. Preparing a matrix solution: respectively adding 8 g of sodium alginate, 4 g of carboxymethyl chitosan, 0.2 g of collagen, 0.72g of berberine hydrochloride and 60 mL of glycerol into 740 m1 of water for dissolving, standing for 12 h at normal temperature for removing bubbles in the solution, and preparing 4 parts of matrix solution.

Example 5

1. Preparing a matrix solution: respectively adding 8 g of sodium alginate, 4 g of carboxymethyl chitosan, 0.2 g of collagen, 1.08 g of berberine hydrochloride and 60 mL of glycerol into 740 m1 of water for dissolving, standing for 12 h at normal temperature for removing bubbles in the solution, and preparing 4 parts of matrix solution.

Example 6

2. And (3) crosslinking: preparing 60 mL of 2% calcium chloride solution, slowly dripping the calcium chloride aqueous solution into the matrix solution by using a rubber head dropper, and slowly stirring to perform a crosslinking reaction.

3. The phenomenon is as follows: the cross-linking agent is not titrated for half, the matrix solution is colloidal, the film agent is difficult to form, and the preparation process condition is not met.

Example 7

1. Preparing a matrix solution: respectively adding 3 g of sodium alginate, 3 g of carboxymethyl chitosan, 0.15 g of collagen and 45 mL of glycerol into 555 m1 of water for dissolving, standing for 12 hours at normal temperature for removing bubbles in the solution, and preparing 3 parts of matrix solution.

2. And (3) crosslinking: preparing 30mL of 2% calcium chloride solution, slowly dripping the calcium chloride aqueous solution into the matrix solution by using a rubber head dropper, and slowly stirring to perform a crosslinking reaction. Titration was stopped before the matrix solution appeared to be gelatinous. Slowly pouring the matrix solution into an aluminum alloy vessel, standing for 15 min, transferring to a 37 ℃ thermostat, and standing for 48 h.

3. The phenomenon is as follows: the formed film agent material is prepared, but the surface of the material has a large amount of bubbles, the thickness distribution is uneven, and the preparation process condition is not met.

Example 8

1. Preparing a matrix solution: respectively adding 3 g of sodium alginate, 6g of carboxymethyl chitosan, 0.15 g of collagen and 45 mL of glycerol into 555 m1 of water for dissolving, standing for 12 hours at normal temperature for removing bubbles in the solution, and preparing 3 parts of matrix solution.

3. The phenomenon is as follows: the prepared film agent material has poor mechanical property, is easy to damage in post treatment and does not accord with the stability of the material.

Test example

1) Material structure and morphology analysis

The products of examples 1-5 were observed by scanning electron microscopy and the results are shown in FIGS. 1-2:

observing the surface morphology of the hemostatic membrane through a scanning electron microscope SEM: firstly, fully drying the hemostatic membrane; secondly, spraying gold for 30 min in a gold spraying chamber; and finally, observing the surface appearance of the sample at the working voltage of 10.0 kV.

The berberine hydrochloride-free product prepared by the scheme in the embodiment 1 has high density, a smooth and flat structure and an obvious fold structure, and is shown in a figure 1; the content of berberine hydrochloride in the products prepared by the schemes of examples 2 to 5 is increased in sequence, and the hemostatic membrane is highly compact and low in light transmittance as a whole. The surface layer of the material forms regular folds and has good adhesion. With the increase of berberine hydrochloride content (fig. 2A-B), the surface gradually tends to be smooth and flat, the gap of folds is reduced, and the distribution density is increased, while when the berberine hydrochloride content is too high (fig. 2C-D), the surface folds are disturbed, even the surface of the material is damaged, so as to cause chaps.

) Swelling Rate analysis

Swelling ratio test experiments were performed on the products of examples 1 to 5, and the results are shown in the following table.

Firstly, weighing the mass (W, unit g) of a centrifugal tube; secondly, adding a small disc material with the diameter of 13 mm into the centrifuge tube, and weighing the total mass (Wi, unit g) of the material and the centrifuge tube; then, adding 1.5 mL of sterile PBS preheated at 37 ℃ into each sample, placing the sample in a constant-temperature water bath kettle at 37 ℃, centrifugally sucking supernatant after swelling the hemostatic membrane every 10 min, draining, weighing the total weight (Ws, unit g), replenishing the original solution again, repeating the steps until the material is completely swelled and the weight is not changed obviously any more, and calculating the Swelling Ratio (SR) of each sample:

the result shows that the water absorption performance of the hemostatic membrane is weaker than that of the positive control. The main reasons are that the commercialized alginate hemostatic dressing and 10% berberine hemostatic microspheres have higher porosity, and the moisture content of the microspheres is very low, so the water absorption rate is higher, and for the hemostatic membrane agent, the material has higher water absorption swelling rate; in addition, the hemostatic material containing berberine hydrochloride has better swelling capacity and water absorption performance. In practical application, the hemostatic effect of the hemostatic material is directly related to the swelling capacity, and the water absorption performance can affect the aggregation of platelets and blood coagulation factors, thereby affecting the hemostatic effect of the material.

2) Analysis of degradation experiments

The degradation rate measurement experiments were conducted on the products of examples 1 to 5, and the results are shown in the following table.

The time required for each sample to reach the maximum swelling ratio can be known from the swelling ratio measurement experiment, the weight (W, unit g) of the centrifuge tube corresponding to each sample is weighed again, and the sample is swelled to the maximum by a small disc sample with the diameter of 13 mm under the water bath at 37 ℃. Each sample was centrifuged at 1500 rpm for 2 min, the PBS buffer was discarded, and the maximum swollen sample and the total mass of the centrifuge tube (Wms, units g) were weighed and recorded. 1.5 mL of 37 ℃ preheated sterile PBS was added to each centrifuge tube, inverted several times upside down to bring the PBS sufficiently into contact with the surface of the sample, and allowed to stand in a 37 ℃ constant temperature water bath. Every 5 min, PBS was removed from the sample and weight recorded (Wt, unit g). Finally, 1.5 mL of fresh 37 ℃ pre-heated PBS was added to the samples, the above steps were repeated until the samples were completely dissolved or no change in quality was observed, and the Degradation Rate (DR) of each sample was calculated:

the result shows that compared with the commercialized alginate hemostatic dressing and 10% berberine hemostatic microspheres, the hemostatic membrane has more excellent degradation capability, can be rapidly degraded, meets the requirement of the degradation performance of the hemostatic material, and particularly meets the requirement of extremely rapid degradation in minimally invasive hemostatic application.

) Analysis of bacteriostatic experiments

The purpose is as follows: the antibacterial activity of the berberine-containing hemostatic microspheres is evaluated by the inhibition zone detection.

The method comprises the following steps: the strains of the experiment are respectively escherichia coli and staphylococcus aureus, and a pre-added bacterium liquid pouring plate method is adopted. Firstly, pouring about 10 mL of sterile LB solid medium into a sterilized clean glass culture dish, and forming a sterile bottom layer after the sterile LB solid medium is coagulated; secondly, taking 10 mL of LB agar medium at 45 +/-2 ℃, and mixing the culture solution: medium = 1:100, 100 μ L of a proper amount of a bacterial solution (1 × 108 CFU/mL) was added, the mixture was gently shaken to distribute the bacteria uniformly, 5 to 10 mL of a solid LB medium containing the bacteria was poured into each dish, and the mixture was allowed to stand at room temperature and cooled and coagulated to form an upper layer containing the bacteria. (the prepared plate should be used as soon as possible and can be stored in a refrigerator at 4 ℃ for a short time.) the bacteriostatic effect of the hemostatic material in wound hemostasis is simulated, and the hemostatic membrane (examples 1-5) is punched into a circular material with the diameter of 6 mm by a puncher. And (3) adding a small paper sheet with the diameter of 6 mm soaked by 0.1 mg/mL ampicillin (Amp +) solution as a positive control into a prepared flat plate, measuring the diameter of the inhibition zone after 12 hours, measuring three groups in parallel, and calculating the average width of the inhibition zone.

Similarly, small paper sheets with the diameter of 6 mm, which are respectively soaked by 10 mg/mL sterile aqueous solution of a hemostatic membrane sample and 0.1 mg/mL ampicillin (Amp +) solution, are added into a prepared flat plate, the diameter of an inhibition zone is measured after 12 hours, three groups are measured in parallel, and the average width of the inhibition zone is calculated, wherein the calculation formula is as follows:

H=(D-d)/2

in the formula: h is the width of the bacteriostatic zone, D is the outer diameter of the bacteriostatic zone, D is the diameter of the sample, and the unit is mm.

In addition, long-term observation is respectively carried out on the bacteriostatic effect directly covered by the hemostatic membrane sample, and the bacteriostatic circle width can be maintained within the observation time of 96 h.

TABLE 1 antibacterial hemostatic membrane antibacterial activity (unit is mm, strain is Staphylococcus aureus)

TABLE 2 antibacterial hemostatic membrane antibacterial activity as a function of time (unit is mm, strain is Staphylococcus aureus)

As a result: as can be seen from the bacteriostatic properties in Table 1, the drug-containing groups all have good effect of inhibiting the growth of Staphylococcus aureus, but have general effect of inhibiting the growth of Escherichia coli (the material itself does not grow bacteria, and has no obvious bacteriostatic zone). In wound hemostasis, example 3 can achieve a more efficient bacteriostatic effect by virtue of excellent swelling and degrading ability. In wound healing, example 4 has a higher berberine hydrochloride content, resulting in a better anti-infective effect.

) Rat tail-cutting hemostasis experiment

The purpose is as follows: the hemostatic effect of the antibacterial hemostatic membrane on the wound surface of rat tail truncation is observed and evaluated.

The method comprises the following steps: the SD rats are randomly divided into a blank control group (without hemostatic materials), a drug-free control group (example 1), a drug-containing experimental group (example 2-5) and two positive control groups (alginate hemostatic dressings and standard gauze), 6 rats (plus, 200-300 g) in each group are subjected to intraperitoneal injection anesthesia with chloral hydrate, 1/2 is cut off from the tail of the rat, the size of the wound surface is basically kept consistent, normal bleeding is guaranteed after natural bleeding for 15 s, the severed wound surface is stretched into a centrifuge tube containing the hemostatic materials, and the bleeding condition of the wound surface is observed at intervals. The time to hemostasis and the amount of blood lost from the rats were recorded and the adhesion of the hemostatic material to the wound surface was observed.

As a result: as can be seen from the data in Table 2, compared with the blank control group, the positive control group and the drug-free control group, the rats containing the drug group have the advantages of obviously shortened hemostasis time, obviously reduced blood loss and good hemostasis effect.

TABLE 2 hemostatic antibacterial film in SD rat tail-cutting hemostatic test results (

, n = 6）

Example 5 shows on the time of hemostasis and the amount of blood loss, and proves that the material is the material with the best hemostatic effect in the experiment. However, we observed in the experiments that after a gush of blood was contacted with an individual experimental group, material breakage occurred, resulting in blood leakage. This may be related to the mechanical properties of the material, making example 5 susceptible to damage during application, such as the increase of berberine hydrochloride content, forming ionic bonds with carboxyl groups in other three types of materials, occupying Ca²⁺And thereby reducing the degree of crosslinking and ductility of the material itself.

According to the rat tail-cutting hemostasis experimental data and the combination of the hemostasis effect and the stability of practical application, the embodiment 3 and the embodiment 4 are relatively ideal hemostasis materials, and compared with a positive control group, the hemostasis speed can be obviously improved; example 5 has excellent hemostatic effect, and attempts have been made to solve the problems of easy chapping and poor stability by improving mechanical properties or compounding with other materials.

) Haemolysis rate analysis of blood cells

The purpose is as follows: evaluation of the hemocompatibility of materials by in vitro hemolysis experiments

The method comprises the following steps: new Zealand white rabbits (female parent/male parent = 1:1, 2-2.5 kg), 10 mL of white rabbit whole blood was drawn from the ear vein of the rabbit. Stirring the blood slowly with glass rod at constant speed for 10 min at room temperature to remove fibrinogen and obtain defibrinated blood. Then, it was placed in a heparin anticoagulant tube, centrifuged at 1500 rpm at 4 ℃ for 10 min, and the supernatant was removed to obtain a high-concentration Red Blood Cell Solution (RBCs). RBCs were suspended in 10 volumes of sterile physiological saline (SPSS), mixed well and then centrifuged at 1500 rpm for 15 min at 4 ℃. The whole process was repeated 3 times and the washed RBCs were resuspended in SPSS to obtain a 2% suspension of RBCs.

First, 0.05 g of alginate hemostatic dressing was soaked in 5 mL of SPSS, left to stand in a 37 ℃ incubator for 24 hours, and then the insoluble material was removed by low-speed centrifugation to obtain a leachate. Secondly, taking 3 parts of 0.5 mL of alginate hemostatic dressing extract to perform a hemolysis experiment, and storing the rest in a refrigerator at 4 ℃; again, 0.05 g of the hemostatic membranes of examples 1-5 were dissolved in 5 mL of SPSS, and 3 parts of 0.5 mL of each sample solution were used for hemolysis experiment. Another 3 tubes of 0.5 mL SPSS and 3 tubes of 0.5 mL ddH were prepared as negative controls₂O as a positive control;

then, putting all samples to be detected together in a constant-temperature water bath kettle at 37 ℃, preheating for 30 min, adding 0.5 mL of 2% RBCs suspension into each tube, and standing the mixed solution in a constant-temperature box at 37 ℃ for 3 h; finally, all sample tubes were centrifuged at 1500 rpm for 10 min, and the OD of the supernatant at 562 nm was measured with an ultraviolet spectrophotometer to calculate the Hemolysis Rate (HR). The calculation formula is as follows (Abs is absorbance):

TABLE 3 hemolytic rate of antibacterial hemostatic membrane

As a result: as can be seen from the data in Table 3, the haemoglobin haemolysis of the alginate haemostatic dressing leachate and the haemostasis solutions of the examples 1-5 is not obvious, and the haemolysis rate is far lower than 5%, which all accord with the pharmacopoeia standard of 2015 edition of the people's republic of China. The antibacterial hemostatic film and the alginate hemostatic dressing are not hemolytic and have good blood compatibility.

) Cytotoxicity assessment

The purpose is as follows: the biocompatibility of the antibacterial hemostatic membrane is evaluated by detecting the relative proliferation rate of L6 cells through an MTT (methyl thiazolyl tetrazolium) experiment

The method comprises the following steps: first, L6 cells in a good state were cultured in advance, and after counting the cells, 100. mu.L of a cell suspension at an appropriate concentration was added to each well, and the cell suspension was uniformly plated in a 96-well plate. And secondly, after culturing for 24 hours in a cell culture box, discarding the original DMEM culture medium, fully and uniformly mixing the prepared solution to be detected and the fresh DMEM culture medium, and adding the solution to the corresponding pore plates to ensure that the concentrations of the added samples in the cell sap are 0.2 mg/mL, 1 mg/mL and 5 mg/mL respectively. Each test sample (examples 1 to 5) and blank (equal amount of sterile PBS buffer) were subjected to 4 duplicate wells of 100. mu.L each and left to stand in a 37 ℃ cell incubator for 48 hours. Then, 4 h before the end of the culture time, the 96-well plate is taken out, the culture solution is discarded, PBS is added into each well for washing once, after the PBS is discarded, 100 mu L of the culture solution and 15 mu L of 5 mg/ml MTT are added, the mixture is gently shaken and uniformly mixed, and the culture is continued for 4 h. After the incubation was completed, the culture medium was discarded, 150. mu.L of DMSO was added to each well, and the mixture was quickly shaken until no precipitate was present in each well. Finally, a set of wells containing only equal amounts of DMSO was added as a zero setting well, absorbance was measured at 490 nm, and the results were recorded and the relative cell proliferation rate (RGR) was calculated as follows:

as a result: the drug-free hemostatic membrane (example 1) itself has no cytotoxicity, but berberine as the antibacterial component of the drug-loaded material containing berberine hydrochloride (examples 2-5) has certain cytotoxicity, just as the cytotoxicity increases with the increase of the content of berberine hydrochloride in the sample. On the one hand, however, the safety of berberine hydrochloride as a medicament has been proved; on the other hand, the drug-containing hemostatic membrane still has excellent cell compatibility (RGR > 65%) at an extremely high concentration of 5 mg/mL, confirming that examples 1-5 can be used as a safe hemostatic.

In summary, the results of comparing the comprehensive properties of the embodiments 1 to 5 show that the hemostatic membrane structure prepared by using 2g of sodium alginate, 1 g of carboxymethyl chitosan, 0.05 g of collagen and 0.09g of berberine hydrochloride has better comprehensive properties. Example 3 can rapidly stop bleeding, has excellent biocompatibility and excellent antibacterial effect, can be used as a novel hemostatic, and is suitable for various kinds of bleeding caused by minimally invasive surgery and daily minor trauma.

7) The change of the viscosity of the antibacterial hemostatic membrane along with the temperature

From fig. 5, it can be observed that the sample of example 5 has the maximum viscosity, and the later gradual decrease shows that the higher the berberine concentration, the higher the viscosity of the antibacterial hemostatic membrane is. Furthermore, with a constant increase in temperature, the viscosity of all samples decreases, possibly with a decrease in the local crosslinking strength of the material due to an increase in the temperature of the sample.

8) The storage modulus G' of the antibacterial hemostatic membrane changes along with the strain

As is apparent from FIG. 6, examples 1 to 5 all showed a decrease in storage modulus with an increase in stress, which is probably due to the fact that the three-dimensional crosslinked structure of the sample was broken under a strong stress, resulting in a gradual decrease in storage modulus. In addition, it can be seen that, under 5% stress, the higher the berberine concentration, the higher the G', the better the elastic deformation ability of the membrane; however, with the increase of stress, the membrane with higher berberine concentration is more likely to be broken, so that the elastic deformation capability of the membrane is rapidly reduced, which indicates that the increase of berberine concentration can improve the crosslinking strength, but the crosslinking structure is more likely to be damaged by stress.

Claims

1. An antibacterial hemostatic membrane comprises sodium alginate, carboxymethyl chitosan and collagen, and is characterized in that the average thickness of the membrane is 0.1-2 mm.

2. The antibacterial hemostatic membrane according to claim 1, wherein the membrane further comprises berberine hydrochloride.

3. The antibacterial hemostatic membrane according to any one of claims 1-2, wherein the mass ratio of sodium alginate to carboxymethyl chitosan to collagen is: 2-3: 0.1-1: 0.05-0.5; or the mass ratio of the sodium alginate to the carboxymethyl chitosan to the collagen to the berberine hydrochloride is 2-3: 0.1-1: 0.05-0.5: 0.01-0.5.

4. The antibacterial hemostatic membrane according to claim 3, wherein the weight ratio of the carboxymethyl chitosan, the sodium alginate, the collagen and the berberine hydrochloride is as follows: 2:1:0.05: 0.03-0.3.

5. The antimicrobial hemostatic film of any one of claims 1-2 or 3-4, wherein the film further comprises a humectant and a cross-linking agent.

6. The antibacterial hemostatic film according to claim 5, wherein the humectant is glycerin and the cross-linking agent is calcium chloride.

7. A method for preparing the antibacterial hemostatic membrane according to any one of claims 1 to 6, which is characterized by comprising the following steps:

(1) preparing a matrix solution: weighing sodium alginate, carboxymethyl chitosan, collagen and berberine hydrochloride, and adding the components according to the mass-volume ratio of 1: 10-10: stirring the mixture with distilled water at normal temperature by magnetic force to form a gelatinous aqueous solution;

8. The method for preparing an antibacterial hemostatic membrane containing berberine hydrochloride according to claim 7, wherein the cross-linking agent in step (3) is an atomized 2% calcium chloride aqueous solution.

9. Use of the antibacterial hemostatic membrane of any one of claims 1-6 in the preparation of a hemostatic material.